The German published patent application DE 196 34 873 A1 describes an apparatus and a method for discriminating, by a time-resolved fluorescence measurement, at least two differently fluorescing types of molecule groups bound to analyte molecules. Therein a light source for illuminating a sample volume is activated for a time-interval T1, then, after a time-interval T2, a detector is activated for a time T3. Which of the at least two molecule groups are contained in the sample volume is determined from the time-dependence of the detector signals recorded in the time-interval T3.
The U.S. Pat. No. 5,315,993 discloses a probe and an apparatus for monitoring a plurality of parameters in an environment by making use of a luminescence phenomenon. To this end a luminescence dye is illuminated with a plurality of excitation light components, the amplitudes of which are modulated in time at specific modulation frequencies. The luminescence response comprises a plurality of luminescence light components, which show modulations corresponding to the modulations of the excitation light. Spectral data resulting from a Fourier-transformation enter model equations from which, inter alia, the life-time of individual luminescence light components can be determined.
The German published patent application DE 101 52 994 A1 describes a method for simultaneously optically determining pH-value and oxygen in solution for a mainly aqueous sample. Therein a single sensor matrix is used, which contains at least two indicator dyes providing at least one distinguishable optical signal for the quantities pH-value and oxygen in solution. In one disclosed embodiment of the method pH-value and oxygen in solution are determined by measuring the relaxation time of a fluorescence response of the indicators following a pulsed excitation.
The European patent application EP 0 442 060 A2 relates to a ratiometric luminescence measurement for determining a variable, for example the concentration of a substance. To this end a first luminescent material with a first absorption band and a second luminescent material with a second absorption band are used; the first and second absorption band do not overlap completely. In alternating first and second illumination intervals the luminescent materials are illuminated with a first excitation light within the first, but outside of the second absorption band, and with a second excitation light within the second, but outside of the first absorption band, respectively. The corresponding luminescence responses of the first and second luminescent material, detected during first and second response intervals, respectively, are evaluated and used for determining the variable.
The article “Luminescence Lifetime Imaging of Oxygen, pH, and Carbon Dioxide Distribution Using Optical Sensors” by G. Liebsch, I. Klimant, B. Frank, G. Holst, and O. S. Wolfbeis in Applied Spectroscopy 54, number 4 (2000), pages 548 to 559, describes the determination of various variables for samples in the wells of a microtiter plate via the dependence of the fluorescence life-time of materials used as sensor on the respective variable. The fluorescence life-time is found as follows: the fluorescence is excited by a light pulse, after the end of which the fluorescence response of the sensors is respectively integrated over two temporally distant intervals of preferentially same duration. The fluorescence life-time is determined from the ratio of the integral values thus obtained. Compared with methods based solely on intensity, this ratiometric method, based on a ratio of measured quantities, has the advantage of being practically independent of local absolute values of the excitation energy.
The article “Fluorescent Imaging of pH with Optical Sensors Using Time Domain Dual Lifetime Referencing” by G. Liebsch, I. Klimant, C. Krause, and O. S. Wolfbeis in Analytical Chemistry Vol. 73, No. 17, Sep. 1, 2001, pages 4354 to 4363, relates to the determination of the pH-distribution in microtiter plates and on a surface. A combination of two luminescent materials, where the ratio of the amounts of the materials is fixed, is used: one fluorescent material, the fluorescence decay-time of which depends on the pH-value, and one phosphorescent material, the phosphorescence decay-time of which is independent of the pH-value. The luminescent materials are excited by illumination, and during the excitation, within a first interval, the combined fluorescence and phosphorescence response of the materials is integrated. Immediately after the end of the excitation the recording of the luminescence response of the materials is interrupted for a period of time which is long enough for the fluorescence to decay practically completely. Afterwards, during a second time interval, which preferentially is of equal length to the first interval, the phosphorescence response of the phosphorescent material is integrated. From the ratio of the two values of the integrals eventually the pH-value can be inferred.
The German published patent application DE 10 2011 055 272 A1 describes a method for determining at least one parameter of a system, wherein the at least one parameter depends on at least one relaxation time of the system. The system is excited by a first sequence of electromagnetic excitation pulses, the sequence having a first defined time gap between consecutive excitation pulses. The response of the system to the first sequence of excitation pulses is integrated continuously over time, and in this way a first response-signal is generated. Likewise, by continuous integration over time of at least a second response of the system a second response-signal is generated. The at least one parameter is determined taking into account the first response-signal and the at least one second response-signal. Preferentially this involves the formation of a ratio of the first and the at least one second response-signal. The system may comprise an object and a sensor means having at least one relaxation time depending on a variable of the object. The parameter, and thus, as the case may be, the variable, may be determined in a space-resolved manner.
Luminescence-based measuring methods are known for the detection and the quantitative determination of many analytes. If the method is based on the intensity of the luminescence phenomenon, a reproducible illumination of the sample studied, in case of the illumination of an area for an extended sample also the spatial homogeneity of the illumination, is crucial. Other methods are based on the decay-time of the luminescence phenomenon, and exploit the fact that this decay-time in case of numerous luminescent materials depends on specific variables of the environment; examples of such variables are pH-value, concentration of a substance, or temperature. With these methods, for which the prior art cited above contains examples, the luminescence response of a substance used as a sensor material is integrated over defined time intervals, and a ratio of the values of the integrals thus obtained is formed. By this formation of a ratio, due to which the methods are classified as ratiometric, the dependence on fluctuations of the illumination is considerably reduced. With these methods it is not necessarily the decay-time or relaxation time of the luminescence phenomenon which is determined explicitly, but instead often a parameter which depends on the relaxation time, for example the ratio of the mentioned values of integrals. If a respective variable to be determined is calibrated against a corresponding respective parameter, the value of the variable can be found from the luminescence response. A difficulty with these methods, however, is to implement the defined time intervals for the integration of the luminescence response with sufficient precision in the measurement apparatus. This involves a certain technical effort implying corresponding costs. Furthermore the technology used is very sensitive, which makes its use, in particular for portable devices in the field, problematic, in particular again with respect to costs.